U.S. patent number 5,019,556 [Application Number 07/177,942] was granted by the patent office on 1991-05-28 for inhibitors of angiogenin.
This patent grant is currently assigned to President and Fellows of Harvard College. Invention is credited to Robert Shapiro, Bert L. Vallee.
United States Patent |
5,019,556 |
Shapiro , et al. |
May 28, 1991 |
Inhibitors of angiogenin
Abstract
An inhibitor of angiogenin and method of use of the inhibitor,
wherein the inhibitor inhibits the angiogenic activity of
angiogenin. The inhibitor is dispensed at a suitable concentration
within a physiologically compatable medium, suitable for
administration to an animal in sufficient quantity to inhibit the
biological activity of naturally occurring angiogenin within the
animal.
Inventors: |
Shapiro; Robert (Holliston,
MA), Vallee; Bert L. (Brookline, MA) |
Assignee: |
President and Fellows of Harvard
College (Cambridge, MA)
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Family
ID: |
27365321 |
Appl.
No.: |
07/177,942 |
Filed: |
April 5, 1988 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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38250 |
Apr 14, 1987 |
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38008 |
Apr 14, 1987 |
4966964 |
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Current U.S.
Class: |
514/13.3;
514/19.3; 530/328; 530/851; 424/583; 530/327; 530/350 |
Current CPC
Class: |
A61P
35/00 (20180101); C07K 14/4703 (20130101); C07K
14/515 (20130101); A61K 38/00 (20130101); Y10S
530/851 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/515 (20060101); C07K
14/47 (20060101); A61K 38/00 (20060101); C07K
015/06 (); C07K 015/12 (); A61K 037/02 (); A61K
035/50 () |
Field of
Search: |
;514/2,21
;424/95,101,105,583 ;530/851,327,328 |
References Cited
[Referenced By]
U.S. Patent Documents
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4727137 |
February 1988 |
Vallee et al. |
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Other References
Taylor et al, Nature, vol. 297, May 27, 1982, pp. 307-312. .
Shapiro et al., Biochemistry 25:3527 (6/17/86). .
Fett et al., Biochemistry 24:5480 (9/14/80). .
Turner et al., Biochem. and Biophys. Res. Comm., 114:1154
(8/12/83). .
Blackburn et al., The Enzymes 15:317 (1982). .
Blackburn et al., J. Biol. Chem. 257:316 (1/10/82). .
Burton et al., Int. J. Peptide Protein Res. 16:359 (1980). .
Blackburn et al., J. Biol. Chem. 255:10959 (11/10/80). .
Blackburn et al., J. Biol. Chem. 254:12488 (12/25/79). .
Blackburn, J. Biol. Chem. 254:12484 (12/10/79). .
Blackburn et al., J. Biol. Chem. 252:5904 (8/25/77). .
Roth, Methods in Cancer Res. 3:154 (1967). .
Folkman et al., Science 235:442 (1987). .
Burton et al., 19 Int. J. Peptide Protein Res. 372 (1982)..
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Primary Examiner: Stone; Jacqueline
Attorney, Agent or Firm: Allegretti & Witcoff, Ltd.
Parent Case Text
BACKGROUND OF THE INVENTION
This is a continuation-in-part of our earlier applications U.S.
Ser. No. 038,008, now U.S. Pat. No. 4,966,964, and U.S. Ser. No.
038,250, now abandoned, each of which was filed Apr. 14, 1987, and
is hereby incorporated by reference.
Claims
We claim:
1. A method of inhibiting growth of a tumor neovascularized as a
result of angiogenin production in an animal, comprising
administering an inhibitor of the angiogenic activity of angiogenin
in sufficient quantity to inhibit said angiogenic activity in said
tumor, wherein said inhibitor comprises human placental
ribonuclease inhibitor, or a substantially equivalent protein from
non-placental human tissue or from non-human mammalian tissue.
2. A method of inhibiting disorders associated with
neovascularization as a result of angiogenin production in an
animal, comprising administering an inhibitor of the angiogenic
activity of angiogenin in sufficient quantity to inhibit said
angiogenic activity in said tumor, wherein said inhibitor comprises
human placental ribonuclease inhibitor, an angiogenin inhibitory
segment of human placental ribonuclease inhibitor, or a
substantially equivalent protein from non-placental human tissue or
from non-human mammalian tissue.
3. A therapeutic composition comprising a polypeptide with an amino
acid sequence that is identical to, conservatively substituted
from, or a deletion product of a PRI sequence, wherein said
polypeptide is capable of inhibiting angiogenin activity, said
polypeptide being dispersed within a medium that is physiologically
compatible and suitable for administration to an animal at a
suitable concentration and in sufficient quantity to inhibit said
angiogenic activity of angiogenin within at least a defined area of
said animal.
Description
Angiogenin is a human protein which induces blood vessel formation
(Fett et al., 24 Biochemistry 5480, 1985). This biological activity
is expressed with an amount as low as 35 fmol (using a chick embryo
CAM assay procedure, Id.). Although originally isolated from medium
conditioned by human tumor cells, angiogenin is not tumor specific,
can be found in a variety of other cells and biological fluids, and
most likely plays a role in normal and/or pathological
neovascularization. It has a molecular weight of about 14,400 and
an isoelectric point greater than about pH9.5, Id. Strydom et al.
24 Biochemistry 5486, 1985 also disclose the amino acid sequence of
angiogenin.
Angiogenin is known to have an enzymatic activity. Specifically, it
catalyzes limited cleavage of 28S and 18S rRNA to produce a
specific pattern of products of 100-500 nucleotides in length
(Shapiro et al., 25 Biochemistry 3727, 1986), but it has no
significant ribonuclease activity in standard RNase assays, Id.
SUMMARY OF THE INVENTION
We have discovered that substances inhibiting at least the
angiogenic activity of angiogenin can be used in methods and
compositions for inhibiting tumor growth. Moreover, substances
inhibiting the above described 18S, 28S rRNA-degrading enzymatic
activity of angiogenin are effective tumor suppressants.
Accordingly, the invention features a method of inhibiting growth
of a tumor in an animal, comprising administering an inhibitor of
the angiogenic activity of angiogenin in sufficient quantity to
inhibit the angiogenin activity in the tumor.
In a second aspect, the invention features a method of inhibiting
growth of a tumor in an animal, comprising administering to the
animal an inhibitor of the following specific enzymatic activity:
cleavage of 18S or 28S rRNA to generally yield segments of 100 to
500 bases.
The following are features of preferred embodiments of the above
methods: The inhibitor is able to bind to RNaseA or to angiogenin;
the inhibitor is the complete natural molecule or segment or
derivatives thereof having the ability to inhibit the above
described enzymatic activity, and most preferably comprises a
segment of a specific substance known as human placental RNase
inhibitor (PRI) having the ability to inhibit the above described
enzymatic activity of angiogenin; other proteins analogous to PRI
from other human tissue or from other mammals also can be used; the
inhibitor is administered at 10-10,000 .mu.g/kg body weight of the
animal; and the animal is a human.
In a third aspect, the invention features an inhibitor capable of
inhibiting the angiogenic activity of angiogenin, wherein the
inhibitor is dispersed within a medium that is physiologically
compatible--i.e., suitable for administration to an animal--at a
suitable concentration and in sufficient quantity to inhibit the
angiogenic activity of naturally occurring angiogenin within at
least a defined area of the animal, for example, the area
immediately surrounding a tumor.
In preferred embodiments of this aspect, the inhibitor inhibits the
enzymatic activity of angiogenin; and most preferably the inhibitor
comprises a segment of PRI (or an analogous protein from other
human tissue or from mammalian tissue) having the ability to
inhibit the 18S, 28S rRNA-degrading activity described above.
In a fourth aspect, the invention features a method of inhibiting
disorders associated with neovascularization. The method comprises
administering to an animal an inhibitor of the angiogenic activity
of angiogenin in sufficient quantity to inhibit angiogenic activity
associated with the disorder.
In a fifth aspect, the invention features a therapeutic composition
comprising a polypeptide comprising the amino acid sequence Val
Asn-Pro Ala Leu-Ala-Glu-Leu (Asn-Leu-Arg), Ser-Asn-Glu-Leu
Gly-Asp-Val-Gly, or Trp or Val-Leu Trp-Leu-Ala-Asp-(Gln or
Cys)-Asp-(Lys or Val). In preferred embodiments, a segment of the
polypeptide has angiogenin inhibiting activity.
In a sixth aspect, the invention features engineered nucleic acid
encoding the above amino acid sequences. Engineered nucleic acid is
defined below, briefly it refers to any nucleic acid removed from
its natural environment.
In a seventh aspect, the invention features engineered nucleic acid
encoding a segment of a polypeptide having angiogenin inhibitory
activity. Preferably, the nucleic acid is obtained by screening a
mammalian gene library with a probe corresponding to a segment of a
polypeptide having angiogenin inhibitory activity; the gene library
comprises genomic or cDNA; the polypeptide is human placental
ribonuclease inhibitor; the library comprises human DNA, or human
cDNA; and the segment is ##STR1##
In an eighth aspect, the invention features a method for making a
polypeptide having angiogenin inhibitory activity. The method
comprises expressing engineered nucleic acid encoding the
polypeptide in a host cell. Preferably, the polypeptide is a
segment of human placental ribonuclease inhibitor.
This invention provides a means for preventing or reducing growth
of tumors. The preferred inhibitor, PRI, is active when present in
a slight molar excess over angiogenin and other PRI-binding
proteins in the body fluids, or within a specified local area, such
as that area immediately surrounding a tumor, and thus need only be
provided at very low concentrations.
Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments thereof
and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The Figures will first briefly be described: Drawings
FIG. 1 is a graphical representation of the effect of angiogenin on
inhibition of RNaseA activity toward uridylyl (3', 5') adenosine
(UpA) by PRI (Blackburn, 254 J. Biol. Chem. 13484, 1979) as an
example of an angiogenin inhibitor.
FIG. 2 is a graphical representation of elution of angiogenin (A)
or angiogenin and PRI (B) from an HPLC column.
FIG. 3 shows the nucleotide sequence of PRI cDNA and the translated
amino acid sequence.
STRUCTURE
Angiogenin
Angiogenin is a protein, having activities as described above,
which can be obtained and purified as described by Fett et al.,
supra, and Shapiro et al., supra. Alternatively, angiogenin can be
obtained and purified using recombinant DNA techniques. Kurachi et
al., 24 Biochemistry 5494, 1985, describe the cDNA and gene
encoding for angiogenin; this cDNA or gene can be expressed from a
standard expression vector and the resulting angiogenin protein
purified by standard procedure, e.g., in mammalian, yeast or
various microbial expression systems. Id. at 5498.
Angiogenin Inhibitors
An angiogenin inhibitor is any compound able to inhibit the
angiogenic activity of angiogenin. Preferably the inhibitor of
angiogenin is a protein; most preferably it is a protein capable of
binding to angiogenin and of inhibiting at least its biological
angiogenic activity and preferably also its enzymatic 28S/18S
rRNA-degrading activity. (The enzymatic activity is measured as
described by Shapiro et al., supra. Briefly, 15-20 .mu.g RNA is
incubated with about 1.9 .mu.M angiogenin at 37.degree. C. in
either 30 mM Hepes or 20 mM Tris, pH 7.5, containing 30 mM NaCl in
a total volume of 13.5 .mu.l. The reaction is terminated after
about 90 minutes using 48 .mu.l of a formamide/formaldehyde
reagent, Id.) Preferably, the inhibitor is isolated from mammalian
tissues, most preferably from human placental tissue.
One example of the inhibitor, PRI, can be isolated as described by
Blackburn (254 J. Biol. Chem. 12,484, 1979). Proteins which are
substantially equivalent to PRI, that is they have similar amino
acid compositions, and have similar biological and enzymatic
inhibitory activities, can be found not only in other human tissues
but in other mammalian tissues. For example, the various inhibitors
of RNAse A described by Burton et al., (19 Int. J. Peptide Protein
Res. 372, 1982, herby incorporated by reference) may be suitable in
this invention. These proteins have about 70-80% similarity in
amino acid composition to PRI, as shown in Table 1 (taken from
Burton et al., Id.). Such proteins are also angiogenin inhibitors
for purposes of the invention. Methods for isolating and purifying
these proteins are given below, using standard affinity
chromatography methodology, or recombinant DNA techniques.
TABLE 1 ______________________________________ Amino acid
compositions of various liver RNase inhibitors and human placental
RNase inhibitor Residues/molecule of inhibitor Liver Placenta Amino
acid Beef Mouse Pig Rat Sheep Human
______________________________________ Asx 47 55 47 59 48 47
Threonine 21 22 25 23 19 16 Serine 45 43 43 43 42 45 Glx 62 63 64
60 62 64 Proline 15 16 15 17 17 17 Glycine 47 33 43 33 51 36
Alanine 37 30 35 29 35 34 Valine 24 20 19 22 23 24 Methionine 2 3 2
2 1-2 2-3 Isoleucine 9 13 9 10 10 12 Leucine 91 94 93 89 92 85
Tyrosine 5 5 4 5 4 4 Phenylalanine 4 3 2 5 4 6 Histidine 6 3 9 6 5
6 Lysine 14 21 15 20 15 17 Arginine 19 19 21 20 20 23 1/2 Cystine +
30 26-27 35 31 27 30 cysteine Tryptophan 5 5 5 5 6 5 Total residues
483 475 486 479 482 473 ______________________________________
Alternatively, the gene encoding an inhibitor may be cloned by
standard techniques. Such techniques include purifying the
inhibitor, determining part of its amino acid sequence, creating a
DNA probe capable of coding for this amino acid sequence, and using
the probe to detect recombinant vectors in a cDNA or genomic
library created from, e.g., placental cells. The cloned gene can
then be expressed in any suitable expression vector in a suitable
expression host cell, e.g., bacteria, yeast, or tissue culture
cells. The recombinant inhibitor protein produced by those cells
can then be purified from the culture supernatant or from the
cells.
Suitable oligonucleotide probes for detecting clones expressing
polypeptides according to the invention can be generated from the
nucleic acid sequence given in FIG. 3. using, e.g., fragments of
the FIG. 3 sequence (or the corresponding antisense sequence) that
are at least 10, and preferably at least 16 bases in length. Probes
with minor modifications (e.g. deviation at less than 3 of 16
positions) of the FIG. 3 sequence are acceptable. The following
fragments are provided by way of example only; the corresponding
antisense strands would be operative also:
5'-TGGCTGTGGCTGGCCGACCAGGAGAA-3'
5'-GTGCTCTGGTTGGCCGACTGCGAT-3'
5'-GTGAACCCTGCCCTGGCTGA-3'
5'-GTGAACCCTGGACTGGCAGA-3'
5'-AATGAGCTGGGCGATGTGGG-3'
5'-AACGAGCTGGGCGATGTCGG-3'
More generalized strands and corresponding antisense strands are:
##STR2## In each of the above sequences B is C or T; I is inosine
(which can base pair with A, T, or C) N is A, T, C or G, and C is C
or I.
Inosine is used in the probe sequence in place of A, T, and C so
that the number of probes to be synthesized as a mixture can be
minimized without prejudicing their hybridizing ability to the
natural gene or cDNA sequences.
One example of a method to isolate a cDNA clone of the gene
encoding PRI is to use the above three antisense strand
oligonucleotide probes under stringent hybridization conditions to
probe a cDNA library of human placental DNA.
Another example of a procedure for isolating and screening cDNA
clones expressing PRI is as follows. Antibodies are raised in
rabbits against PRI purified as described above. The antibodies are
purified by affinity chromatography, using PRI coupled to an
activated solid matrix such as Sepharose (e.g. activated by
CNBr).
Any clones which hybridize to one or more of the probes, and
preferably hybridize to all three, or any clones that express
proteins that bind to anti PRI antibodies, are potential clones of
the PRI-encoding gene. To determine whether the clones do encode
PRI, they can be purified, followed by re-checking of
re-hybridization with the probes or of expression of proteins that
bind anti-PRI antibodies.
Ultimately, the clones can be sequenced and the sequences compared
to see if they correlate with the known properties of PRI, i.e.,
the amino acid sequence, molecular weight and amino acid
composition. Expression of PRI can then be achieved by insertion of
the cDNA clone into an expression vector and the recombinant PRI
which is expressed, isolated, and purified. All such clones contain
engineered DNA, that is, DNA taken from its natural environment and
inserted into a vector, such as a plasmid or phage, or even within
the genome of an organism. Thus, the engineered DNA is no longer
surrounded by naturally occuring sequences on either side of it.
Generally such engineered DNA is constructed using recombinant DNA
techniques, and does not include naturally occurring DNA in which,
for example, a translocation of chromosomal DNA in vivo has changed
the environment of DNA; however it does include such natural events
which occur after the DNA has been manipulated in some way by
recombinant DNA methodology.
We have determined the nucleotide sequence of human PRI cDNA and
the amino acid sequence of human PRI inferred from that nucleotide
sequence. We have confirmed a portion of that amino acid sequence
by Edman degradation of PRI tryptic peptides.
FIG. 3 shows the PRI cDNA sequence and translated amino acid
sequence. A bacterium, E. coli DH5.alpha., transformed with vector
pUC18-PRI(A) containing the cDNA of FIG. 3 has been deposited with
the American Type Culture Collection in Maryland under access
number 67668 on Mar. 31, 1988.
Angiogenin inhibitors can be obtained using the PRI information in
FIG. 3, e.g., by producing recombinant PRI inhibitor using standard
techniques for culturing cells that include the PRI cDNA on a
suitable expression vector, and purifying the PRI from the culture
supernatant.
Fragments and variants of the PRI of FIG. 3 sequence are also
suitable. For example, PRI can be fragmented (e.g. by tryptic
digestion) and the resulting fragments (purified by HPLC) can be
assayed as described elsewhere in this application for angiogen
inhibitory activity. Specific PRI fragments of use are as follows:
##STR3##
PRI from other mammals, such as the ones shown in Table 1, can be
obtained and analyzed by cloning them from other mammals and
providing angiogenic fragments as described above.
Once cloned, the naturally occurring gene may also be modified by
standard techniques, for example, by altering the amino acid
sequence of the inhibitor, so long as the inhibitor retains the
ability to inhibit the biological angiogenic activity of
angiogenin. Such alterations may be designed to increase the
binding ability of the inhibitor for angiogenin. Further, related
inhibitor-encoding genes can be isolated by using a part of the
above described cloned gene encoding an inhibitor as a probe for
libraries, constructed by standard techniques, of other animal
genomes.
In order to isolate other inhibitors, one possible procedure
includes detecting proteins able to bind angiogenin, for example,
by binding angiogenin to a column and passing a sample containing
potential inhibitor proteins through this column. (RNaseA can be
used in place of angiogenin, as described by Blackburn 254 J. Biol.
Chem. 12488, 1979.) Bound protein can then be eluted, for example,
by using 0.1 M sodium acetate pH 5.0 containing 3 M NaCl, 15%
glycerol, 1 mM EDTA and 5 mM DTT, which is able to dissociate the
angiogenin/inhibitor complex. The eluted protein can then be
purified and tested, as described below, to see if the released
proteins inhibit the angiogenic activity of angiogenin, or
alternatively if they inhibit its enzymatic activity. The active
protein(s) may then be purified by standard procedure.
Further, it is possible to use segments of inhibitors, such as
segments of PRI, which retain the biological inhibiting character
of the native inhibitor. Such segments can be created by standard
procedure using proteases to digest natural inhibitors to produce
active segments; or by using recombinant DNA techniques to remove
non-essential parts of the structural gene or cDNA encoding the
inhibitor, and then expressing this engineered DNA to produce a
modified inhibitor.
Inhibitory Activity
Below is an example of in vitro measurements relative to the
inhibitory effect of PRI on angiogenin. This example is not meant
to be limiting for the invention and those skilled in the art will
realize that these examples are indicative of whether other
inhibitors or segments thereof can be used in the methods described
below.
Measurement of Inhibition of the Enzymatic Activity of
Angiogenin
Angiogenin and PRI were purified generally as described by Fett et
al. and Shapiro et al., cited above. The enzymatic activity of
angiogenin was measured as described by Shapiro et al., supra.
Briefly, angiogenin catalyzes the cleavage of 28S and 18S rRNA to
form products of 100 to 500 nucleotides in length. These products
are stable and include a large, approximately 500 bases, segment
which is visible in an agarose gel. In one experiment, 12 .mu.g of
RNA was incubated with or without angiogenin and/or PRI at
37.degree. C. in 33 mM Hepes, and 33 mM NaCl, pH7.5. After 30 min
the reaction was terminated, as described by Shapiro et al., supra,
the samples run on a 1.1% agarose gel, and stained with ethidium
bromide. 0.96 .mu.M PRI completely inhibited the enzymatic
degradation of rRNA by 0.8 .mu.M angiogenin. PRI alone had no
activity on rRNA. Thus, PRI is a potent inhibitor of the enzymatic
activity of angiogenin, being active at only just over a 1:1 ratio
with angiogenin.
Stability of the Angiogenin/PRI Complex
For therapeutic use of the angiogenin inhibitor it is advantageous
to use an inhibitor that binds strongly to angiogenin and reduces
its biological activity. PRI is such an inhibitor.
RNaseA also binds to PRI and competes with angiogenin for binding
PRI in solution. The enzymatic activity of RNaseA is also inhibited
by PRI. These properties can be used to determine the stability of
the PRI/angiogenin complex, as follows.
UpA is an excellent substrate for RNaseA but not for angiogenin. In
one experiment, a varying amount of angiogenin was added to a
mixture of 0.27 nM RNaseA and 0.20 nM PRI, with the RNaseA and
angiogenin being mixed together first, followed by PRI for 5 min.
at 25.degree. C., and then the UpA substrate (0.2 mM, in a buffer
of 10 .mu.g/ml human serum albumin (HSA), 0.1 M 2-(N
morpholino)-ethane-sulfonic acid, 0.1 M NaCl, and 1 mM EDTA pH6.0).
The more angiogenin present in the mixture the more PRI that will
be bound to it, and thus the less PRI that will be bound to RNaseA
and able to inhibit its enzymatic activity. Further, the stronger
the binding of PRI to angiogenin than to RNaseA, the greater the
reduction in inhibition of RNaseA activity, since PRI will then
preferentially bind to angiogenin. The results are shown in FIG. 1.
Relative inhibition is calculated as (Vo-Va)/(Vo-Vr) where Vo
denotes velocity of RNaseA activity in the absence of PRI, Vr
denotes velocity in the presence of PRI with no angiogenin added,
and Va is velocity in the presence of PRI with angiogenin added. At
5.8 nM angiogenin there is essentially no inhibition of RNaseA
activity. Thus, a large excess of angiogenin is needed to remove
all the PRI from reacting with and inhibiting the RNaseA.
In a related experiment, the Ki for angiogenin and PRI can be
estimated using the above method and modifying it so that the PRI
and angiogenin are firstly preincubated for 10 minutes, followed by
addition of RNaseA and UpA. This assay determines the level of free
inhibitor remaining after incubation of PRI and angiogenin. Thus,
if the dissociation of PRI and angiogenin is slow there will be
little inhibition of RNaseA activity. At a ratio of angiogenin:PRI
of 1:1.2 no free PRI was detected. Thus, PRI and angiogenin are
tightly bound and appear to have a Ki less than 0.1 nM.
The strength of binding of PRI and angiogenin was also demonstrated
by cation exchange HPLC. This process can distinguish free
angiogenin from angiogenin/PRI complex. A Synchropak CM 300 column
(250 .times.4.1 mm; Sychrom, Inc.), a Waters Associates liquid
chromatography system and a Hewlett-Packard 3390A integrator were
used; the results are shown in FIG. 2. Samples of 1 ml in 0.1 M
Tris, pH7, containing 1 mM EDTA and 10 .mu.g HSA were eluted with a
10 min. linear gradient from 220-620 mM NaCl in 20 mM sodium
phosphate, pH7, at a flow rate of 1 ml/min.; effluents were
monitored at 214 nm. In panel A is shown elution of 0.64 .mu.g
angiogenin; in panel B the elution of 0.64 .mu.g angiogenin and 12
.mu.g PRI. There is no detectable free angiogenin shown in panel B.
Addition of RNaseA to the angiogenin - PRI mixture at 32-fold
excess did not produce free angiogenin, even after 17 h incubation.
Thus, dissociation of angiogenin/PRI appears to have a half life of
greater than 1 day.
Inhibition of Angiogenic Activity
Angiogenesis (i.e., angiogenic activity) was assessed by a
modification of the chick embryo chorioallantoic membrane (CAM)
assay of Knighton et al. 35 Brit. J. Cancer 347 (1977) described by
Fett et al., supra. For this assay PRI was desalted using an Amicon
Centricon-10 microconcentrator to dilute the buffer at least 500
fold. This removes interfering components, from the PRI solution,
which may adversely affect the experiment or may harm the egg
itself. The results of adding PRI to the angiogenin are shown in
Table 2 below.
Data for experiments #1 and #2 represent composites of results
obtained in 2 and 3 sets of assays, respectively. Between 10 and 20
eggs were utilized for each of the three experimental groups within
each set. The following amounts of angiogenin and PRI were
employed: Experiment #1, 75 ng and 5 2 .mu.g, respectively;
Experiment #2, 46 ng and 700 ng; and Experiment #3, 25 ng and 180
ng.
TABLE 2 ______________________________________ % Positives (Total
Number of Eggs) Experiment Sample #1 #2 #3
______________________________________ Angiogenin 58 (24) 52 (54)
62 (13) PRI 17 (29) 33 (48) 17 (12) Angiogenin + PRI 15 (26) 25
(52) 7 (14) ______________________________________
From these results it is evident that excess PRI decreases
angiogenin activity to a level indistinguishable from that observed
with the buffer and inhibitor alone controls.
Inhibition of Tumor Growth
For immunotherapeutic studies, male nude (nu/nu) mice (Charles
River Laboratories) were maintained under laminar flow conditions,
and were age- and cage matched (5 animals per cage) prior to
experimentation. Experimental animals (5-10 per group) were
injected subcutaneously (S.C.) with 5 .times.10.sup.5 HT 29 human
colon adenocarcinoma cells (Fogh et al., In Human Tumor Cells In
Vitro, pp115-160 ed. Fogh, Plenum Press, New York., 1975) on day 0.
Also on day 0, animals were treated with either buffer control (the
buffer used to store PRI dialysed against phosphate buffered
saline; 0.2 g/l KCl, 0.2 g/l KH 8 g/l NaCl and 2.16 g/l Na.sub.2
HPO.sub.4.7H.sub.2 O, pH7.4 at 37.degree. C., PBSA 100 .mu.l) or
placental inhibitor (100 .mu.l) at varying dosages by S.C. or
intraperitoneal (I.P.) injection. Treatment regimens included daily
injections (10-11 doses) or injections at 2 3 day intervals (5-10
doses) depending on the experimental protocol. Assessment of animal
health, weight, tumor size as well as photographic records were
recorded 2-3 times per week. At the termination of the experiments
blood and tissue samples were collected for immunological and
histological evaluation.
In one experiment, forty male nu/nu mice were injected S.C. with 5
.times.10.sup.5 HT-29 cells on day 0. Additionally, on day 0 mice
1-10 (Group 1) received 100 .mu.l of control buffer; mice 11-20
(Group 2) received 10 .mu.g in 100 .mu.l of placental inhibitor;
mice 21-30 (Group received 1.0 .mu.g in 100 .mu.l of placental
inhibitor; and mice 31-40 received 0.1 .mu.g in 100 .mu.l of
placental inhibitor. Groups 1 and 2 received further injections of
buffer or 10 .mu.g of placental inhibitor, respectively, on days 1,
4, 6, 8, 18, 20, and 22. On days 1, 4, 6 and 8, Groups 3 and 4
received placental inhibitor at dosages of 1.0 .mu.g or 0.1 .mu.g,
respectively. All treatment injections were given I.P. After 60
days, only animals 3, 14, 17, 18, 20 and 24 remained tumor free.
Thus, inoculation with 10 .mu.g PRI prevented tumor formation in
about 60% of animals.
Inhibition of both the biological and enzymatic activities of
angiogenin by an inhibitor has important mechanistic, physiologic
and pharmacologic implications. It is consistent with the
hypothesis that these two actions of angiogenin are interrelated,
as suggested previously by the simultaneous loss of both activities
upon carboxymethylation by bromoacetate at pH 5.5. Shapiro et al.,
supra. Further it raises the possibility that such inhibitors may
play a role in the in vivo regulation of angiogenin. The
angiogenin/PRI interaction likely involves regions of angiogenin
separated widely in the three-dimensional structure, many of them
outside the active center. Thus, conservation of residues necessary
for enzymatic activity alone probably cannot account for the
strength of the interaction.
This implies that the capacity of angiogenin to bind PRI has been
maintained independently during evolution. Apparently, binding by
PRI and other inhibitors reflects a physiologically relevant
control mechanism having pharmacologic and therapeutic potential.
For example, as shown above, these inhibitors are active in
prevention of tumor formation in mice. Apparently they are suitable
for injection into humans or other animals in order to prevent
tumor growth, and other diseases associated with
neovascularization, e.g., diabetic retinopathy, rheumatoid
arthritis, and Kaposi's sarcoma. That is, they are suitable for
treating disorders where vascularization plays an important role in
the pathophysiology of diseases such as solid tumors, hemangiomata
and psoriasis.
Preferably, they are injected intravascularly, most preferably
intravenously, or injected intraperitoneally in a physiologically
compatible medium (such as PBSA, or those buffers and agents
described by Krakoft, Ca-A Cancer Journal for Clinicians,
March/April 1987, Vol. 37:93), suitable for administration to an
animal in a sufficient quantity, e.g., about 10-10,000 .mu.g/kg
body weight of the animal, to inhibit naturally occuring biological
activity of angiogenin within the animal. Alternatively, they can
be administered topically to a specific area, or injected
subcutaneously.
We have found that these inhibitors are stable for at least 2 weeks
even at concentrations as low as 8 .mu.M when they are in a
purified form; there is no need to include DTT in the storage
buffer or the therapeutic composition being administered, however,
natural agents, which are physiologically compatible, e.g., N
acetyl cysteine or cysteine or cysteamine may be used. For
administration it is important that the buffer fluid not injure the
patient. It is possible that these inhibitors may be used
prophylatically at a low concentration. Thus, for example, it is
appropriate to administer 10-10,000 .mu.g/kg body weight to
patients at risk of cancer development, e.g., patients who have had
a primary tumor removed.
Other embodiments are within the following claims.
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